Non-Phosphodiester Polynucleotide Analogs
DNA consists of covalently linked units, each composed of a nucleobase (adenine, cytosine, guanine, or thymine) attached to a pentose sugar (deoxyribose) via a glycosidic linkage, with a phosphate ester (phosphodiester) linking successive sugar rings. Numerous types of DNA analogs have been synthesized, with most variations typically having a modification or replacement of the phosphodiester backbone.
Examples of non-phosphodiester polynucleotide analogs having a modified phosphate backbone include: methylphosphonates, phosphorothioates, phosphoramidites, phosphorodithioates, phosphorotriesters, and boranophosphates. An alternative approach is the development of structural mimetics of the phosphodiester linkage, generally with the objective of providing a backbone linkage that is charge neutral (to increase the stability of DNA hybrid complexes), relatively hydrophobic (to increase cellular uptake), and achiral. Examples of non-phosphodiester polynucleotide analogs wherein the phosphodiester backbone is replaced by a structural mimic linkage include: alkanes, ethers, thioethers, amines, ketones, formacetals, thioformacetals, amides, carbamates, ureas, hydroxylamines, sulfamates, sulfamides, sulfones, glycinylamides, and others.
In addition to replacing the phosphodiester linkage, alternative approaches have replaced the entire (deoxy)ribose-phosphate backbone, retaining just the nucleobases. One of these approaches replaces the entire (deoxy)ribose-phosphate backbone with a peptide-like backbone, generating a so-called "peptide nucleic acid", "polyamide nucleic acid", or simply "PNA" (Nielsen et al. (1991) Science 254: 1497; Nielsen et al. (1994) Bioconj. Chem. 5: 3; Leijon et al. (1994) Biochemistry 33: 9820; Huang et al. (1991) J. Org. Chem. 56: 6007; Egholm et al. (1993) Nature 365: 556; Buchardt et al. (1993) Trends Biotechnol. 11; 384; Nielsen PE (1995) Rev Biophys Biomol Struct 24: 167;Agrawal et al. (1995) Curr Opin Biotechnol. 6: 12; Nielsen et al. (1993) Anticancer Drug Res. 8: 53; Cook PD (1991) Anticancer Drug Des. 6: 585, incorporated herein by reference). PNAs have an achiral, noncharged backbone, as exemplified by a backbone composed of N-(2-aminoethyl)glycine units, which is a suitable structural mimic of DNA. Hybrids between such a PNA and complementary sequence DNA or RNA are reported to exhibit higher thermal stability per base pair than DNA:DNA or RNA:RNA duplexes (Wittung et al. Nature 368: 561).
PNAs have been reported to have many interesting properties. Binding of PNA to double-stranded DNA occurs by strand invasion via formation of a D-loop strand displacement complexes (Egholm et al. (1993) Nature 365: 556) that have unique biological properties, including the capacity to serve as artificial transcription promoters in some contexts (Mollegaard et al. (1994) Proc. Natl. Acad. Sci. (U.S.A.) 91: 3892). PNAs have been shown to bind to DNA and RNA in a sequence-dependent manner (Brown et al. (1994) Science 265: 777; Egholm et al. (1993) op.cit), and exhibit superior base pair mismatch discrimination in PNA/DNA hybrids than do DNA/DNA duplexes (Orum et al. (1993) Nucleic Acids Res 21: 5332).
PNAs have been used to target the single strand-specific nuclease S1 to a PNA/DNA hybrid formed via strand invasion, making S1 nuclease act like a pseudo restriction enzyme (Demidov et al. (1993) Nucleic Acids Res 21: 2103). Alternatively, complementary PNAs have been used to block sequence-specific DNA restriction enzyme cleavage of dsDNA plasmids (Nielsen et al. (1993) Nucleic Acids Res 21: 197). PNAs have been used to arrest transcription elongation by targeting a complementary sequence PNA to the template DNA strand (Nielsen et al. (1994) Gene 149: 139). PNA strand invasion has also been shown to inhibit transcriptional activation by the transcription factor NF-.kappa.B by blocking its interaction with 5' regulatory sequences to which it normally binds (Vickers et al. (1995) Nucleic Acids Res 23: 3003).
Interaction of certain DNA-binding ligands with PNA/DNA 1:5 hybrids has also been reported (Wittung et al. (1994) Nucleic Acids Res 22: 5371).
The antisense and antigene properties of PNAs have been reported (Bonham et al. (1995) Nucleic Acids Res 23: 1197; Hanvey et al. (1992) Science 258: 1481; Nielsen et al. (1993) AntiCancer Drug Des 8: 53). A vector-mediated delivery method for introducing PNAs through phospholipid membranes and through the blood-brain barrier have been reported (Pardridge et al. (1995) Proc. Natl. Acad. Sci. (U.S.A.) 92: 5592; Wittung et al. (1995) FEBS Lett 375: 27). Orum et al. (1995) Biotechniques 19: 472 report a method for sequence-specific purification of nucleic acids by PNA-controlled hybrid selection.